Therapeutic proteins have been used in the medical field for more than 100 years and are constantly gaining in medical interest and significance. Comparisons with other active pharmaceutical substances show that therapeutic proteins as albuminous substances exhibit a significantly lower stability and a higher degree of impurities. In comparison with other pharmaceutical drug substances, therapeutic proteins also exhibit a relatively high sensitivity to various chemical and physical factors, in particular to enzymes auch as proteases that cleave peptide bonds. Therefore, all measures which, during the recovery, purification and storage of intact therapeutic proteins, contribute to maintaining their integrity and stability are of great economic importance.
Changes in therapeutic proteins—and that applies to all proteins—may be effected by intramolecular rearrangements or chemical reactions such as the cleavage or formation of covalent bonds. An enzyme which recognizes a therapeutic protein as its substrate is able to largely change the same and, as far as a protease is concerned, may result in one or more cleavages in the peptide chain of the protein. Such enzymes as well as their precursors, which are referred to as proenzymes or—as in the case of proteases—also as zymogens, can already be contained in the starting materials for the production of natural therapeutic proteins as well as those obtained by genetic engineering. Enzymes which act upon proteins are proteins themselves in most cases and can be provided both in the free state and as already bound to their substrates. Zymogens may also be provided as substrates or may already be bound to their specific enzymes which they convert to proteases.
Enzymatic effects on therapeutic proteins during their manufacture and storage, ranging from the starting product to the final product, may lead to high losses and instable final products (Anderson et al. 1986; Eaton et al. 1986; Brummel et al. 1999).
Enzymes acting upon therapeutic proteins as well as their proenzymes and procofactors, which, by activation, lead to the formation of further such enzymes during the purification procedure, are already present in many starting materials. Procofactors also are proteins which are disintegrated into cofactors by the effect of proteases without being enzymes themselves. Cofactors specifically increase certain protease activities many times over.
Such enzymes, proenzymes, cofactors or procofactors may themselves be utilized as therapeutic proteins, provided that viral pathogens as they can be present in blood or in biomasses obtained by genetic engineering were inactived or depleted, respectively, by inactivation or cleansing processes in the course of preparing the pharmaceutically active substance.
The blood clotting cascade is one of the best known enzyme systems. It is triggered by an enzyme cofactor, the lipid-containing tissue factor, which increases the enzyme activity of Clotting Factor VIIa many times over. The tissue factor normally is found only in cells and therefore cannot enter a relationship of interaction with Clotting Factor VIIa. Due to pathologic or traumatic events, the tissue factor may reach the surface of tissue factor-containing cells or may leave those cells, respectively. By means of the small amounts of Factor VIIa which always are present in the blood, the enzyme cascade may then be initiated. The final point of that clotting cascade is the enzyme thrombin which is formed in this way via the extrinsic and common pathway and transforms fibrinogen to fibrin. Fibrinogen, which may also be used as a therapeutic protein, is cleaved into fibrin monomer and fibrinopeptides by thrombin. Fibrin monomer polymerizes into fibrin strands and finally into a network which may bring about a closure of the wound in the wound bed and hence the termination of bleeding. Fibrinogen neither is an enzyme nor does it possess any cofactor properties (Lawson et al. 1994; Hemker 2002; Mann et al. 2002).
However, thrombin does not only have an enzymatic effect on fibrinogen but, among other things, it also converts the proenzyme of Clotting Factor XIII to the enzyme of Clotting Factor XIIIa. By means of cross linkages between the fibrin strands in the three-dimensional fibrin scaffold, said enzyme acting as transglutaminase gives rise to an increased biomechanical stability of the fibrin network that was formed as well as to a protection from enzymatic degradation. In the blood or plasma, respectively, which has clotted due to the effect of thrombin, further important enzymatic processes arise, in particular the formation of thrombin from the proenzyme prothrombin, the Clotting Factor II. That brings about a strong increase in the thrombin concentration in the clotted blood, which does not only cause the complete conversion of Clotting Factor XIII to that of XIIIa, but also the activation of proenzyme TAFI into enzyme TAFIa. Said enzyme cleaves off a short peptide from the fibrin, which short peptide contains receptors for a fibrinolytic enzyme complex and thus serves for the resistance of fibrin to any fibrinolytic effects. For those processes, a relatively high concentration of thrombin in the already clotted blood is necessary, which may be generated by activating the prothrombin present in the clotted blood (Siebenlist et al. 2001).
The strongly increased formation of thrombin in the blood clot itself is no longer accomplished via the extrinsic tenase pathway but via the intrinsic tenase and the common pathways. Small amounts of thrombin in the presence of Factor VIII and Factor IXa and of any clotting-promoting phospholipid already lead to the formation of the intrinsic tenase complex, which, just like the extrinsic tenase complex but 50 times stronger, causes the activation of Clotting Factor X in Xa by proteolytic cleavage, which then leads to the formation of thrombin by the activation of prothrombin (Mutch et al. 2001).
In physiological as well as in pathophysiological clotting processes, certain cellular phospholipid structures in and on cells, in particular in blood platelets, trigger a strong additional acceleration of certain enzymatic processes in the clotting cascade, in addition to the activity-increasing, high-molecular cofactors.
Already in the hitherto known methods for the recovery of therapeutic proteins, purification conditions were applied which endeavoured to minimize the enzymatic effects on the proteins to be obtained, for instance, by carrying out the purification process at temperatures as low as possible and/or by the addition of inhibitors (Kunicki et al. 1987 and 1992; Johnson et al. 1994 and 1996; Rotblat et al. 1985). Nevertheless said procedure did not turn out to be sufficient, in particular in case of proteins which are subject to easy enzymatic cleavage. The cleansing of such enzymes, its proenzymes, cofactors and procofactors also was not done sufficiently enough in quantitative terms during the purification process and hence the reproduction of such enzymes throughout the entire manufacturing process was not taken into sufficient account, either. For that reason, many of the hitherto obtained therapeutic proteins were characterized by merely an insufficient yield with changed and decreased biological efficiency as well as great instability, in particular during the necessary storage of the finished drug composition.
The enzyme inhibitors that have so far been used for purification exhibited an avidity and/or a concentration which were too low. Therefore, enzymes which were already bound to their substrates could be neutralized only insufficiently. Furthermore, it also was not taken into account to keep the concentration of enzyme inhibitors as constant as possible during the purification process.
It is possible that therapeutic proteins obtained by genetic engineering may already have bound proteolytic enzymes when being expressed and discharged from the cells, or they may bind those, respectively, which are present in the culture medium. The same also applies to therapeutic plasma proteins. During the recovery, storage and processing of plasma, especially proteolytic enzymes which recognize plasma proteins as their substrates can be formed. In doing so, at first protease-substrate complexes are formed and, in the following, the substrate is enzymatically cleaved.
After that kind of complex formation involving a therapeutic protein as a high-molecular substrate, proteases are no longer or only to a minor extent inhibitable by inhibitors. By those processes, therapeutic proteins may undergo a substantial change in quality or be largely destroyed already during their manufacture and especially their storage.
Already in the first fractionation process of the cryoprecipitation of plasma, enzyme-mediated changes in the cryoprecipitate and in the cryoprecipitate supernatant may occur, resulting in a poorer yield and a reduction in quality of the therapeutic proteins obtained at a later stage of the manufacturing process. This applies in particular to Clotting Factor VIII obtainable from cryoprecipitate. Enzymatic processes in the cryoprecipitate may also lead to the activation of Clotting Factor XIII, which, together with thrombin, is responsible for the increased formation of fibrin monomer complexes. Fibrin monomer complexes have an adverse effect on the quality of the fibrinogen obtainable from cryoprecipitate.
A mixture of clotting factors can be eluted from the supernatant of the cryoprecipitation by binding to weak anion exchangers and elution with saline solutions. Said mixture contains basically Clotting Factors II, VII, IX and X and is referred to as a prothrombin complex. It is also possible to isolate individual clotting factors from the prothrombin complex, processing them further into a pharmaceutical preparation. The prothrombin complex, processed further into a pharmaceutical preparation, must pass through further manufacturing steps in order to guarantee an extensive removal of possibly present viruses. These manufacturing steps must be carried out at room temperature or at an increased room temperature. Due to the starting material and the production, there is the possibility that clotting factors present in the prothrombin complex are activated, which, in the parenteral application of the prothrombin complex, are at least also involved in side effects of said product which may manifest themselves as peripheral occlusions, myocardial infarctions, pulmonary embolisms or strokes. Therefore, the object is to obtain the clotting factors contained in the prothrombin complex in such a way that they are activated neither during storage, nor during the manufacturing process, nor during the subsequent application.
Activations of zymogens play an adverse role also during the recovery of thrombin, plasminogen and immunoglobulins obtained from different fractions of the cryoprecipitation supernatant.
Thrombin can be contaminated, for instance, by plasmin evolving from its zymogen, plasminogen, and therefore might exhibit insufficient haemostatic activity. According to the plasmin content, the result is a premature loosening of the wound closure and a reoccurrence of bleeding.
Activations of zymogens also play an important role in the recovery of immunoglobulins. By plasmin, immunoglobulin G is cleaved, for example, into a Fab-fragment and a Fc-piece. If the immunoglobulin G is applied parenterally, this enzymatic cleavage of the immunoglobulin leads to a strongly reduced half-life and to a reduction in quality of the antibodies contained in the immunoglobulin preparation. For therapeutic purposes, it is often necessary to administer large amounts of immunoglobulin G—up to 250 g per patient. In this connection, severe side effects have repeatedly been reported, such as myocardial infarctions and central vascular occlusions. It therefore is of utmost importance to produce immunoglobulin preparations which do not contain any procoagulants.
The protease effect may often result in a homomeric and heteromeric formation of aggregates, which may even cause a visible formation of particles from insoluble aggregates, increases in viscosity, a deceleration of the necessary filtration steps, decreased yields and an unsatisfactory stability during the storage of intermediate and finished products.
The problem arises from the necessity to prevent, as far as possible, the formation of proteases and their effects on therapeutic proteins during the manufacture and storage thereof so that unsatisfactory yields during the necessary purification processes as well as reductions in quality as a result of changes in their molecular structure caused by enzymatic processes are avoided. Therefore, it is necessary to implement manufacturing processes that allow for a recovery and reprocessing of the starting material without resulting in enzyme-mediated changes in the therapeutic proteins during the required purification process, including the removal of viruses.
Thus, it is necessary that, during manufacture and storage, the therapeutic proteins are protected by an appropriate cleansing and/or the addition of enzyme inhibitors from all direct effects of enzymes which recognize them as substrates as well as from any indirect effects caused by proenzymes, cofactors and procofactors.
It is also necessary to determine the enzyme concentrations which do not yet lead to modifications in a particular therapeutic protein, taking into consideration the exposure time and other conditions important for the enzymatic activity.
For the stability of therapeutic proteins, it is necessary to measure enzyme activities over lengthy periods of time in order to be able to infer the long-term stability from the accelerated stability test. Such accelerated stability tests usually last for a period of at least one month.
Since even very small enzyme amounts may lead to modifications in therapeutic proteins, the development of appropriate determination methods lasting for fairly long amounts of time is necessary.
It is also important to measure the inhibitor amount which is required for inhibiting unremovable proteases and for maintaining said inhibition in the long run.